I propose to study the process of mixed-function oxidation as it occurs in oxidative demethylation and deformylation of 4-methyl and 4-formyl sterols. Specifically, my study will have four objectives. 1. I will study the topology of the microsomal membrane as it relates to oxidative demethylation and deformylation. To do this I will investigate the effect of protease treatment of microsomal vesicles on oxidative activity. 2. I will study the origin of NADH and NADPH both of which provide electrons for oxidative demethylation and deformylation. In particular, I will investigate the manner in which these cofactors arise through microsomally associated reactions. I will attempt to isolate, purify and characterize with regard to function and membrane-protein interaction a NADP ion-protein which appears to bind NADP ion and serves as a source for conversion to NADPH by both microsomal and extramicrosomal dehydrogenases. Further, I will attempt the same studies on a microsomal dehydrogenase which converts NAD ion to NADH. 3. The cofactors NADH and NADPH will be studied further by attempting to determine if microsomally produced cofactors are compartmentalized and thus will not equilibrate between the microsomes and extramicrosomal space. 4. Finally, I will attempt to determine whether NADH and NADPH produced in the microsomes are available as electron donors for other systems in addition to oxidation demethylation and deformylation of methyl and formyl sterols.